The intricate network between the p34 and p44 subunits is central to the activity of the transcription/DNA repair factor TFIIH.

The general transcription factor IIH (TFIIH) is a multi-protein complex and its 10 subunits are engaged in an intricate protein-protein interaction network critical for the regulation of its transcription and DNA repair activities that are so far little understood on a molecular level. In this study, we focused on the p44 and the p34 subunits, which are central for the structural integrity of core-TFIIH. We solved crystal structures of a complex formed by the p34 N-terminal vWA and p44 C-terminal zinc binding domains from Chaetomium thermophilum and from Homo sapiens. Intriguingly, our functional analyses clearly revealed the presence of a second interface located in the C-terminal zinc binding region of p34, which can rescue a disrupted interaction between the p34 vWA and the p44 RING domain. In addition, we demonstrate that the C-terminal zinc binding domain of p34 assumes a central role with respect to the stability and function of TFIIH. Our data reveal a redundant interaction network within core-TFIIH, which may serve to minimize the susceptibility to mutational impairment. This provides first insights why so far no mutations in the p34 or p44 TFIIH-core subunits have been identified that would lead to the hallmark nucleotide excision repair syndromes xeroderma pigmentosum or trichothiodystrophy.

ARCH domain of XPD, an anchoring platform for CAK that conditions TFIIH DNA repair and transcription activities.

The xeroderma pigmentosum group D (XPD) helicase is a subunit of transcription/DNA repair factor, transcription factor II H (TFIIH) that catalyzes the unwinding of a damaged DNA duplex during nucleotide excision repair. Apart from two canonical helicase domains, XPD is composed of a 4Fe-S cluster domain involved in DNA damage recognition and a module of uncharacterized function termed the "ARCH domain." By investigating the consequences of a mutation found in a patient with trichothiodystrophy, we show that the ARCH domain is critical for the recruitment of the cyclin-dependent kinase (CDK)-activating kinase (CAK) complex. Indeed, this mutation not only affects the interaction with the MAT1 CAK subunit, thereby decreasing the in vitro basal transcription activity of TFIIH itself and impeding the efficient recruitment of the transcription machinery on the promoter of an activated gene, but also impairs the DNA unwinding activity of XPD and the nucleotide excision repair activity of TFIIH. We further demonstrate the role of CAK in downregulating the XPD helicase activity within TFIIH. Taken together, our results identify the ARCH domain of XPD as a platform for the recruitment of CAK and as a potential molecular switch that might control TFIIH composition and play a key role in the conversion of TFIIH from a factor active in transcription to a factor involved in DNA repair.

Identification of the binding site for the regulatory calcium-binding domain in the catalytic domain of NOX5.

NADPH oxidases (NOX) are important superoxide producing enzymes that regulate a variety of physiological and pathological processes such as bacteria killing, angiogenesis, sperm-oocyte fusion, and oxygen sensing. NOX5 is a member of the NOX family but distinct from the others by the fact that it contains a long N-terminus with four EF-hand Ca(2+)-binding sites (NOX5-EF). NOX5 generates superoxide in response to intracellular Ca(2+) elevation in vivo and in a cell-free system. Previously, we have shown that the regulatory N-terminal EF-hand domain interacts directly and in a Ca(2+)-dependent manner with the catalytic C-terminal catalytic dehydrogenase domain (CDHD) of the enzyme, leading to its activation. Here we have characterized the interaction site for the regulatory NOX5-EF in the catalytic CDHD of NOX5 using cloned fragments and synthetic peptides of the CDHD. The interaction was monitored with pull-down techniques, cross-linking experiments, tryptophan fluorescence, hydrophobic exposure, isothermal titration calorimetry, and cell-free system enzymatic assays. This site is composed of two short segments: the 637-660 segment, referred to as the regulatory EF-hand-binding domain (REFBD), and the 489-505 segment, previously identified as the phosphorylation region (PhosR). NOX5-EF binds to these two segments in a Ca(2+)-dependent way, and the superoxide generation by NOX5 depends on this interaction. Controlled proteolysis suggests that the REFBD is autoinhibitory and inhibition is relieved by NOX5-EF.

Scherffelia dubia centrin exhibits a specific mechanism for Ca(2+)-controlled target binding.

Centrins are calcium binding proteins that belong to the EF-hand (or calmodulin) superfamily, which are highly conserved among eukaryotes. Herein, we report the molecular features and binding properties of the green alga Scherffelia dubia centrin (SdCen), a member of the Chlamydomonas reinhardtii centrin (CrCen) subfamily. The Ca(2+) binding capacity of SdCen and its isolated N- and C-terminal domains (N-SdCen and C-SdCen, respectively) was investigated using flow dialysis and isothermal titration calorimetry. In contrast with human centrin 1 and 2 (from the same subfamily), but like CrCen, SdCen exhibits three physiologically significant Ca(2+) binding sites, two in the N-terminal domain and one in the C-terminal domain. Mg(2+) ions could compete with Ca(2+) in one of the N-terminal sites. When Ca(2+) binds, the N-terminal domain becomes more stable and exposes a significant hydrophobic surface that binds hydrophobic fluorescent probes. The Ca(2+) binding properties and the metal ion-induced structural changes in the C-terminal domain are comparable to those of human centrins. We used isothermal titration calorimetry to quantify the binding of SdCen, N-SdCen, and C-SdCen to three types of natural target peptides, derived from the human XPC protein (P17-XPC), the human Sfi1 protein (R17-hSfi1), and the yeast Kar1 protein (P19-Kar1). The three peptides possess the complete (P17-XPC and R17-hSfi1) or partial (P19-Kar1) centrin binding motif (W(1)L(4)L(8)). The integral SdCen exhibits two binding sites for each target peptide, with distinct affinities for each site and each peptide. The high-affinity peptide binding site corresponds to the C-terminal domain of SdCen and displays binding constants and the poor Ca(2+) sensitivities similar to those observed for human centrins. The low-affinity site constituted by the N-terminal domain is active only in the presence of Ca(2+). The thermodynamic binding parameters suggest that the C-terminal domain of SdCen may be constitutively bound to a target, while the N-terminal domain could bind a target only after a Ca(2+) signal. SdCen is also able to interact with calmodulin binding peptides (W(1)F(5)V(8)F(14) motif) with a 1:1 stoichiometry, whereas the isolated N- and C-terminal domains have a much lower affinity. These data suggest particular molecular mechanisms used by SdCen (and probably by other algal centrins) to respond to cellular Ca(2+) signals.

Psychological Determinants of Investor Motivation in Social Media-Based Crowdfunding Projects: A Systematic Review.

Background: Using the power of Internet, crowdfunding platforms are currently changing the traditional landscape of fundraising. Social media-based IT platforms in particular are bringing the creators of crowdfunding projects closer than ever to potential investors. A large variety of factors function as determinants of individuals' intention to participate in crowdfunding and have an intertwined impact on funding as the ultimate project goal. Objectives: For a better understanding of investor behavior in social media-based crowdfunding projects, this paper covers identifying, analyzing, and classifying general and specific factors of investor motivation, based on the literature in the field.The main focus is the relationship between the affordances provided by social media-based crowdfunding platforms and the psychological determinants of investor motivation in innovative start-up projects. Methods: Using IEEE Explore, Clarivate Web of Science, ScienceDirect, and Scopus, we conducted a systematic review of the existing research on the emerging role of crowdfunding as a disruptive technology in financing the start-up innovative projects. The paper explores the main determinants of investor motivation and aims to streamline the success factors in crowdfunding campaigns. Results: A total of 1,216 publications were identified after searching the aforementioned databases and, upon refining the results, 515 articles were considered for the final sample. After reading the titles and abstracts, the sample was reduced to 78 articles that were read in-depth and synthesized in accordance with the defined research questions. The selected articles were clustered into three main categories: general studies, determinants of investor behavior, and success factors. Conclusions: In the new global economy, crowdfunding platforms have become the nexus between the emerging creators of innovative products and services and the necessary funding sources. This connection is possible via a cumulative collection of contributions from multiple investors recruited from the audience of the selected platform, without time or space constraints. However, the determinants of the investment decision are very different in the case of social media-based crowdfunding platforms compared to determinants in the mainstream environment. This paper surveys these motivators and reveals how platform features can be used to persuade individuals to make a financial contribution toward the success of a project.

TFIIH: A multi-subunit complex at the cross-roads of transcription and DNA repair.

Transcription factor IIH (TFIIH) is a multiprotein complex involved in both eukaryotic transcription and DNA repair, revealing a tight connection between these two processes. Composed of 10 subunits, it can be resolved into a 7-subunits core complex with the XPB translocase and the XPD helicase, and the 3-subunits kinase complex CAK, which also exists as a free complex with a distinct function. Initially identified as basal transcription factor, TFIIH also participates in transcription regulation and plays a key role in nucleotide excision repair (NER) for opening DNA at damaged sites, lesion verification and recruitment of additional repair factors. Our understanding of TFIIH function in eukaryotic cells has greatly benefited from studies of the genetic rare diseases xeroderma pigmentosum (XP), Cockayne syndrome (CS) and trichothiodystrophy (TTD), that are not only characterized by cancer and aging predispositions but also by neurological and developmental defects. Although much remains unknown about TFIIH function, significant progresses have been done regarding the structure of the complex, the functions of its catalytic subunits and the multiple roles of the regulatory core-TFIIH subunits. This review provides a non-exhaustive survey of key discoveries on the structure and function of this pivotal factor, which can be considered as a promising target for therapeutic strategies.

Molecular architecture of the SYCP3 fibre and its interaction with DNA.

The synaptonemal complex (SC) keeps homologous chromosomes in close alignment during meiotic recombination. A hallmark of the SC is the presence of its constituent protein SYCP3 on the chromosome axis. During SC assembly, SYCP3 is deposited on both axes of the homologue pair, forming axial elements that fuse into the lateral element (LE) in the tripartite structure of the mature SC. We have used cryo-electron tomography and atomic force microscopy to study the mechanism of assembly and DNA binding of the SYCP3 fibre. We find that the three-dimensional architecture of the fibre is built on a highly irregular arrangement of SYCP3 molecules displaying very limited local geometry. Interaction between SYCP3 molecules is driven by the intrinsically disordered tails of the protein, with no contact between the helical cores, resulting in a flexible fibre assembly. We demonstrate that the SYCP3 fibre can engage in extensive interactions with DNA, indicative of an efficient mechanism for incorporation of DNA within the fibre. Our findings suggest that SYCP3 deposition on the chromosome axis might take place by polymerization into a fibre that is fastened to the chromosome surface via DNA binding.

Cryo-electron tomography of SYCP3 fibers under native conditions.

The synaptonemal complex (SC) forms during the early stages of meiotic prophase I, when it mediates the pairing of homologous chromosomes. Despite the crucial role of the SC in chromosome synapsis and genetic recombination, the molecular details of its function are still unclear. High-resolution information on the structure of SC proteins would be very valuable to elucidate the molecular basis of their function in meiosis. Here we show how cryo-electron tomography and subtomographic averaging can be usefully applied to provide insights into the structure of the helical SYCP3 protein in its filamentous state. The establishment of such method should prove of use for structural studies of other SC proteins, such as SYCP1 and the TEX12-SYCE2 complex, which can form physiologically relevant filamentous assemblies, and ultimately for the structural analysis of the SC.

The production of multiprotein complexes in insect cells using the baculovirus expression system.

The production of a homogeneous protein sample in sufficient quantities is an essential prerequisite not only for structural investigations but represents also a rate-limiting step for many functional studies. In the cell, a large fraction of eukaryotic proteins exists as large multicomponent assemblies with many subunits, which act in concert to catalyze specific activities. Many of these complexes cannot be obtained from endogenous source material, so recombinant expression and reconstitution are then required to overcome this bottleneck. This chapter describes current strategies and protocols for the efficient production of multiprotein complexes in large quantities and of high quality, using the baculovirus/insect cell expression system.

Insect cells-baculovirus system for the production of difficult to express proteins.

The production of sufficient quantities of homogenous protein not only is an essential prelude for structural investigations but also represents a rate-limiting step for many human functional studies. Although technologies for expression of recombinant proteins and complexes have been improved tremendously, in many cases, protein production remains a challenge and can be associated with considerable investment. This chapter describes simple and efficient protocols for expression screening and optimization of protein production in insect cells using the baculovirus expression system. We describe the procedure, starting from the cloning of a gene of interest into an expression transfer baculovirus vector, followed by generation of the recombinant virus by homologous recombination, evaluation of protein expression, and scale-up. Handling of insect cell cultures and preparation of bacmid for co-transfection are also detailed.

Phytochemical and pharmacological studies on Orthosiphon stamineus Benth. (Lamiaceae) hydroalcoholic extracts.

The main components of Orthosiphon stamineus Benth. leaves and extracts are the pharmacologically active polyphenols: the polymethoxylated flavonoids and the caffeic acid derivatives. Two tinctures, having different alcohol concentration, were studied from phytochemical and pharmacological point of view. The main polyphenols were identified and quantitatively determined by HPLC. Comparison of the retention parameters and UV-Vis spectra of standards and those of the separated compounds performed the identification of caffeic-, cichoric- and rosmarinic acids, respectively, of sinesetine and eupatorine. The quantitative determination was performed by external standard method. The diuretic, saluretic and uricosuric actions of the studied tinctures were compared by experiments on rats.

Low HDL-cholesterol: a strong predictor of glycemic response to glitazone treatment in patients with type 2 diabetes.

We performed a study in 102 people with type 2 diabetes aiming to determine "easy-to-use" predictive factors for glycemic response to glitazones. We found that low baseline HDL-cholesterol (<40 mg/L [1.04 mmol/L] in males, <50 mg/L [1.30 mmol/L] in females) was a strong independent predictor of glycemic response to glitazones (OR=2.67 [2.02-3.52], p=0.0004).